Although highly active retroviral therapy (HAART) dramatically reduces viral load and restores CD4 T-cell counts in HIV-infected individuals, its success is hampered by the fact that HIV can establish a latent proviral reservoir within long-lived resting memory CD4 lymphocytes. The molecular underpinnings of HIV latency involve multiple host factors and pathways actively repressing HIV transcription and maintaining the proviral genome in a transcriptionally inert state. Many host factors have been implicated based on genome-wide screens in cell-line based models of latency. But the exact role of these candidate genes in HIV latency occurring within resting primary CD4 T cells, remain unknown. The evaluation of these genes in resting primary CD4 T cells is hampered by the fact that shRNAs cannot be introduced into these cells using lentiviral vectors. In this collaboration among three CARE investigators (Warner C. Greene, Jonathan Karn, Vincente Planelles), we now propose to utilize a novel technology based on HIV-2 Vpx to render resting CD4 T cells permissive to lentivirus infection. This technology will permit efficient knockdown and evaluation of the candidate regulatory genes in resting primary CD4 T cells. This approach first involves pretreating cells with virion like particles (VLP) encapsulating Vpx to remove anti-viral reverse transcription block mediated by SAMHD1 followed by shRNA lentiviral transduction. A number of potential positive and negative regulators of HIV latency identified in CARE-sponsored studies will be systematically tested including the genome organizer and transcription repressor CTCF, select receptor subunits in the immunosuppressive PDCD1 and pro-inflammatory interleukin IL22RA pathways, and a putative Tat cofactor termed TRIM32. The evaluation of these cellular genes in primary CD4 T cell models of HIV latency formed in memory CD4 T cells, Th17 CD4 T cells and Treg CD4 T cells promises to provide key insights into the role of these genes in HIV latency. These studies could lay the foundation for finding small molecules that when combined will promote effective purging of the latent reservoir in patients while not producing a general state of cellular activation.